Analysis of Endocytic Protein Dynamics by Stochastic Modeling of Fluorescent Signal Lifetimes

Single molecule fluorescence microscopy has allowed us to visualize cellular dynamics and organization with more precision than ever before. One such method is SRAP (Single molecule Recovery After Photobleaching) used in the... [ view full abstract ]

Single molecule fluorescence microscopy has allowed us to visualize cellular dynamics and organization with more precision than ever before. One such method is SRAP (Single molecule Recovery After Photobleaching) used in the Berro Lab at Yale University, which is optimized to visualize single molecules in a diffraction-limited spot. That is, the probability of two fluorescently labeled molecules occupying the same spatiotemporal location is very low. Here, we consider a theoretical case where the SRAP method is being used to image monomers in a linear filament, such as actin. In particular, we seek to develop an accurate mathematical model of the fluorescent signal lifetime distributions of monomers in linear filaments. To do so, we developed a stochastic model to express fluorescent lifetimes for increasing numbers of additional monomers within a single linear filament. MATLAB simulations revealed that more additional monomers increases the overall fluorescence signal lifetime of the molecule. The average fluorescence signal lifetime was found to increase significantly for each additional monomer, using perturbative theory to perform the analytical calculations. This theoretical simulation allows scientists to distinguish between distinct molecular events when performing single molecule fluorescence microscopy, which is especially useful for dynamically complex cellular processes such as clathrin-mediated endocytosis.